Summary
[125Iodo]cyanopindolol [(±)-ICYP], a potent and selective ligand for β-adrenoceptors, exhibited complex biphasic dissociation kinetics. Consequently, in receptor binding studies, the pure (+)- and (-)-enantiomers of ICYP were synthesised and their equilibrium and kinetic binding characteristics were investigated on a membrane preparation of guinea pig left ventricle containing almost only β1-adrenoceptors.
All three ligands, i. e. (+)-, (-)- and (±)-ICYP, bind to β-adrenoceptors as assessed by competition experiments with different β-blocking agents; irrespective of the ligand used, the same dissociation constant was found for the competitor. In a first series of saturation binding experiments performed in a low concentration range of free ligand (0–250 pM), ICYP showed the following dissociation constants: K D=93, 9 and 23 pM, and number of binding sites: B max=40,128 and 124 fmoles/mg protein for (+)-, (-)-, and (±)-ICYP, respectively. Asexpected, (±)-ICYP showed the same B max as (-)-ICYP, whereas its K D was approximately two times higher than that of (-)-ICYP. Surprisingly, the B max of (+)-ICYP represented only ∼ 30% of the B max of (-)-ICYP. All three ligands bound apparently to a single class of binding sites.
In dissociation experiments, the enantiomers of ICYP showed biphasic dissociation curves as observed for the racemic ligand. (+)-, (-)- and (±)-ICYP showed a rapidly dissociating (k -1=0.488, 0.047 and 0.049 min−1) and a slowly dissociating component (k -2=0.0205, 0.0033 and 0.0025 min−1). The ratio slow dissociating/fast dissociating component represented respectively for (+)-, (-)- and (±)-ICYP 40/60, 90/10 and 90/10. For all three ligands, the association rate constants were of the same order of magnitude (ca. 109 M−1 min−1), typical for a diffusion controlled reaction.
In contrast to equilibrium binding studies, the existence of multiple receptor affinity sites was evident from the biphasic dissociation behaviour observed especially with the nonracemic ligands (+)-ICYP and (-)-ICYP.
Simulation of theoretical saturation curves performed with the ratios of high versus low affinity sites and the K D-values suggested by kinetic analysis, indicated that the delineation into two affinity states might be visible in saturation experiments, under certain conditions.
Therefore, equilibrium binding studies were repeated with an increased number of ligand concentrations covering a large concentration range of 0–800 pM. Simultaneous analysis of saturation curves from the same experiment using three different ligands, provided more accurate estimates of the ratio of high and low affinity sites, as well as the affinity constants of the ligand for each receptor affinity state, in good agreement with the results from kinetic analysis.
The contribution of the (+)enantiomer in the binding of the racemic ligand under low receptor concentrations could be neglected since dissociation characteristics of (±)- and (-)-ICYP were identical. A model that explains the biphasic dissociation of (±)-ICYP by differential binding of both enantiomers could be rejected. Kinetic and equilibrium binding characteristics of the three radioligands were not influenced by the guanylnucleotide Gpp(NH)p (10−4 M).
The antagonist ICYP binds to β-adrenoceptors in a high and low affinity state which are probably interconvertible.
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Abbreviations
- CYP:
-
cyanopinodolol
- ICYP:
-
[125Iodo]cyanopindolol
- HYP:
-
(±)-hydroxybenzylpindolol
- IHYP:
-
(±)-[125Iodo]hydroxybenzylpindolol
- 3H-DHA:
-
(-)-[3H]dihydroalprenolol
- 3H-HBI:
-
(±)-[3H]-hydroxybenzylisoproterenol
- ISA:
-
intrinsic sympathomimetic activity; Gpp(NH)p, guanyl-5′yl-imidodiphosphate
- FM 24:
-
1-(2-exobicyclo[2,2,1]hept-2yl-phenoxy)-3-[(1-methylethyl)amino]-2-propranolol
References
Boyd ND, Cohen JB (1980) Kinetics of binding of [3H] acetylcholine and [3H]carbamoylcholine to Torpedo postsynaptic membranes: slow conformational transitions of the cholinergic receptor. Biochemistry 19:5344–5353
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248–254
Bürgisser E, Hancock AA, Lefkowitz RJ, De Lean A (1981a) Anomalous equilibrium binding properties of high-affinity racemic radioligands. Mol Pharmacol 19:205–216
Bürgisser E, Lefkowitz RJ, De Lean A (1981b) Alternative explanation for the apparent “two-step” binding kinetics of high affinity racemic antagonist radioligands. Mol Pharmacol 19:509–512
Danilewicz JC, Kemp JEG (1973) Absolute configuration by asymmetric synthesis of (+)-1-(4-Acetamidophenoxy)-3-(isopropylamino)-propan-2-ol (Practolol). J Med Chem 16:168–169
Engel G (1980) Identification of different subgroups of beta-receptors by means of binding studies in guinea-pig and human lung. Triangle 19:(2) 69–76
Engel G, Hoyer D, Berthold R, Wagner H (1981) (±)-[125Iodo]-cyanopindolol, a new ligand for β-adrenoceptors: identification and quantitation of subclasses of β-adrenoceptors in guinea pig. Naunyn-Schmiedeberg's Arch Pharmacol 317:277–285
Fraser RR, Petit MA, Saunders JK (1971) Determination of enantiomeric purity by an optically active nuclear magnetic resonance shift reagent of wide applicability. Chem Commun 1450–1451
Frosst and Co. Belgian Patent No. 733387.
Galper JB, Klein W, Caterall WA (1977) Muscarinic acetylcholine receptors in developing chick heart. J Biol Chem 252:8692–8699
Hedberg A, Minneman KP, Molinoff PB (1980) Differential distribution of beta-1 and beta-2 adrenergic receptors in cat and guinea pig heart. J Pharmacol Exp Ther 212:503–508
Heidenreich KA, Weiland GA, Molinoff PB (1980) Characterization of radiolabeled agonist binding to β-adrenergic receptors in mammalian tissues. J Cyclic Nucl Res 6:217–230
Insel PA, Stoolman LM (1978) Radioligand binding to beta adrenergic receptors of intact cultured S49 cells. Mol Pharmacol 14:549–561
Kent RS, De Lean A, Lefkowitz RJ (1980) A quantitative analysis of beta-adrenergic receptor interactions: resolution of high and low affinity states of the receptor by computer modeling of ligand binding data. Mol Pharmacol 17:14–23
Klein WL (1980) Multiple binding states of muscarinic acetylcholine receptors in membranes from neuroblastoma X glioma hybrid cells. Biochem Biophys Res Commun 93:1058–1066
Le Fur G, Schmelck PH, Geynet P, Hanoune J, Uzan A (1978) Cardiac β-adrenergic receptor: evaluation of FM 24 as a new and very slowly dissociable blocker. Life Sci 23:1841–1850
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Lucas M, Homburger V, Dolphin A, Bockaert J (1979) In vitro and in vivo kinetic analysis of the interaction of a norbornyl derivative of propranolol with β-adrenergic receptors of brain and C6 glioma cells; an irreversible or slowly reversible ligand. Mol Pharmacol 15:588–597
Maclicke A, Fulpius BW, Klett RP, Reich E (1977) Acetylcholine receptor: responses to drug binding. J Biol Chem 252:4811–4830
Maguire ME, Wiklund RA, Anderson HJ, Gilman AG (1976) Binding of [125I]Iodohydroxybenzylpindolol to putative β-adrenergic receptors of rat glioma cells and other cell clones. J Biol Chem 251:1221–1231
Manalan AS, Besch HR Jr, Watanabe AM (1981) Characterization of [3H]-(±)Carazolol binding to β-adrenergic receptors. Circ Res 49:326–336
Michel T, Hoffman BB, Lefkowitz RJ (1982) Regulation of adenylate cyclase by adrenergic receptors. In: Briggs M, Corbin A (eds) Progress in hormone biochemistry and pharmacology, Eden Press, Quebec (in press)
Molinoff PB, Wolfe BR, Weiland GA (1981) Quantitative analysis of drug-receptor interactions: determination of the properties of receptor subtypes. Life Sci 29:427–443
Morris TH, Kaumann AJ (1979) Characteristics of 3H-(±)-carazol binding to myocardial β-adrenoceptors. Naunyn Schmiedeberg's Arch Pharmacol, Suppl 308:R33
Patil PN, Miller DD, Trendelenburg U (1975) Molecular geometry and adrenergic drug activity. Pharmacol Rev 26:323–392
Quast U, Schimerlik M, Lee T, Witzemann V, Blanchard R, Raftery MA (1978) Ligand-induced conformation changes in Torpedo californica membrane-bound acetylcholine receptor. Biochemistry 17:2405–2414
Rodbell M (1980) The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284:17–22
Ross EM, Maguire ME, Sturgill TW, Biltonen RL, Gilman AG (1977) Relationship between the β-adrenergic receptor and adenylate cyclase. J Biol Chem 252:5761–5775
Snedecor GW, Cochran WG (1973) Curvilinear regression. In: Snedecor GW, Cochran WG (eds) Statistical methods, 6th ed. The Iowa State Univ. Press, Iowa/USA, pp 447–471
Verrier B, Fayet G, Lissitzky S (1974) Thyrotropin-binding properties of isolated thyroid cells and their purified plasma membranes. Eur J Biochem 42:355–365
Weiland G, Taylor P (1979) Ligand specificity of state transitions in the cholinergic receptor: behavior of agonists and antagonists. Mol Pharmacol 15:197–212
Weinstock LM, Mulvey DM, Tull R (1976) Synthesis of the β-adrenergic blocking agent Timolol from optically active precursors. J Org Chem 41:3121–3124
Ziegelhoffer A, Anand-Srivastava MB, Khandelwal RL, Dhalla NS (1979) Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase. Biochem Biophys Res Commun 89:1073–1081
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Hoyer, D., Engel, G. & Berthold, R. Binding characteristics of (+)-, (±)- and (-)-[125Iodo] cyanopindolol to guinea-pig left ventricle membranes. Naunyn-Schmiedeberg's Arch. Pharmacol. 318, 319–329 (1982). https://doi.org/10.1007/BF00501172
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DOI: https://doi.org/10.1007/BF00501172